From the early part of this century, viruses have been used to treat cancer. The approach has been two-fold; first, to isolate or generate oncolytic viruses that selectively replicate in and kill neoplastic cells, while sparing normal cells. Investigators initially used wild type viruses, and this approach met with some, albeit, limited success. While oncolysis and slowing of tumor growth occurred with little or no damage to normal tissue, there was no significant alteration in the course of the disease. See, Smith et al., Cancer 9: 1211-1218 (1956), Cassel, W. A. et al., Cancer 18: 863-868 (1965), Webb, H. E. et al., Lancet 1: 1206-1209 (1966). See, also, Kenney, S and Pagano, J. J. Natl. Cancer Inst., vol. 86, no. 16, p.1185 (1994).
More recently, and because of the reoccurrence of disease associated with the limited efficacy of the use of wild type viruses, investigators have resorted to using recombinant viruses that can be delivered at high doses, and that are replication competent in neoplastic but not normal cells. Such viruses are effective oncolytic agents in their own right, and further, can be engineered to carry and express a transgene that enhances the anti neoplastic activity of the virus. An example of this class of viruses is an adenovirus that is mutant in the E1B region of the viral genome. See, U.S. Pat. No. 5,677,178, and Bischoff, J. R., D. H. Kim, A. Williams, C. Heise, S. Horn, M. Muna, L. Ng, J. A. Nye, A. Sampson-Johannes, A. Fattaey, and F. McCormick. 1996, Science. 274:373-6.
The approach described in U.S. Pat. No. 5,677,178, inventor, McCormick takes advantage of the loss of tumor suppressor proteins in cancer cells, particularly p53. A function of p53 is to inhibit the progression of mammalian cells through the cell cycle in response to DNA damage. The E1b-55K protein of wild-type adenovirus binds to p53 in adenovirus infected cells that exhibit p53 and produce a substantial inactivation of p53 function. Functional adenoviral E1b-55K protein is essential for efficient adenoviral replication in cells containing functional p53. Adenovirus mutants which substantially lack the ability to bind p53 are replication deficient in non-replicating, non-neoplastic cells having normal levels of functional p53. However, such adenoviral mutants exhibit a replication phenotype in cells which are deficient in p53 function (for example, cells which are homozygous for substantially deleted p53 alleles, cells which comprise mutant p53 proteins which are essentially non-functional) and thus cause the death of such cells.
There are several reports on adenoviral mutants which substantially lack the ability to bind p53 resulting from a mutation in the E1B-55K protein. Such viruses generally have some, or all of the E1B-55K region deleted.
U.S. Pat. No. 5,677,178, inventor, McCormick, describes, among other things, adenoviral mutants that lack a viral oncoprotein, that is E1B-55K protein or E4 orf6, capable of binding p53.
U.S. Pat. No. 6,080,578 describes, among other things, adenoviral mutants that have deletions in the region of the E1b-55K protein that is responsible for binding p53.
There are several publications which suggest that in certain tumor cells which have p53 that the presence of p53 may not be predictive of the efficacy of an adenovirus that encodes an E1b-55K protein that is defective in binding p53. See, for example, Goodrum and Ornelles J. Virol. 1998, Vol. 72: 9479-9490; Hall, et al., 1998, Nat. Med. 4: 1068-1072; Hay, et al., 1999, Hum., Gene Ther. 10: 579-590; Rothmann, et al., 1998, J. Virol. 72: 9470-9478; and Turnell, et al., 1999, J. Virol. 73: 2074-2083. It is believed that in these cells that there is a defect in the p53 pathway, and thus such cells lack functional p53, and consequently also support replication of adenoviral mutants that encode an E1b-55K protein that is defective in binding p53.
The adenoviral E1B-55K protein plays an important role during the productive infection of human adenovirus type 5 (Ad5). In the early phase of infection, E1B-55K forms a stable complex with p53 (Samow, P., et al., Virology, 1982, vol. 120: p. 510-517), and inhibits p53-mediated transcriptional activation (Yew, P. R. and A. J. Berk, Nature, 1992, vol. 357: p. 82-85; and Yew, P. R., et al., Genes Dev., 1994, vol. 8: 1994). Furthermore, E1B-55K and another adenoviral protein, E4orf6, co-operate to relocate p53 to the cytoplasm for active degradation (Querido, E., et al., J. Virol. 1997, vol. 71: p. 3788-3798; Roth, J. et al., J. Virol. 1998; vol. 72: p. 8510-8516; and Steegenga, W. T., et al., Oncogene, 1998, vol. 16: p. 349-357). This inactivation of p53 is critical for the replication of adenovirus, which requires cells to enter S phase. During the late phase of infection with Ad5, viral mRNAs are selectively exported to the cytoplasm and are efficiently translated, while the nucleocytoplasmic transport of most host cell mRNAs is inhibited (Beltz, G. A. and S. J. Flint, J. Mol. Biol., 1979, vol. 131: p. 353-373; Babiss, L., et al., Mol. Cell. Biol., 1985, vol. 5: p. 2552-2558; Pilder, S., et al., Mol. Cell. Biol., 1986: vol. 6: p. 470-476). This selective accumulation of viral mRNAs during the late phase of infection is mediated by a protein complex that includes E1B-55K and E4orf6 (Sarnow, P., et al., J. Virol., 1984, vol. 49: p. 692-700; and Halbert, et al., J. Virol., 1985, vol. 56: p. 250-257; and, Bridge, E. and G. Ketner, Virology, 1990, vol. 174: p. 345-353) This complex actively shuttles between the nucleus and the cytoplasm, serving as a nucleocytoplasmic transporter for viral mRNAs (Dobbelstein, M. et al., EMBO J., 1997, vol. 16: p. 4267-4284).
In clinical trials that are still ongoing, an adenovirus mutant, ONYX-015, that lacks the ability to bind p53 has been shown to be biologically active and cause partial tumor necrosis in head and neck cancer. ONYX-015, originally named dl 1520, is a mutant adenovirus that does not express the E1B-55K protein (Barker, D. D. and A. J. Berk, Virology, 1987, vol: 156: p. 107-121). This virus contains a stop codon immediately following the translation initiation codon ATG, plus a large deletion of the E1B-55K coding sequence. These mutations result in the complete abrogation of E1B-55K expression but do not alter the expression of the E1B-19K protein encoded in an overlapping open reading frame. Consequently, this virus lacks the ability to bind and inactivate p53, allowing it to replicate substantially selectively in tumor cells that are defective in p53 function but not in normal cells where p53 function is normal (Bischoff, J. R., et al., Science, 1996, vol. 274: p. 373-376; and, Heise, C., et al., Nat. Med., 1997, vol. 3: p. 639-645). This forms the foundation of utilizing ONYX-015 as an anti-tumor agent.
While the lack of E1B-55K function in ONYX-015 permits viral replication in tumor cells that lack p53 function, the virus is also defective in cytoplasmic accumulation of the viral late mRNAs, host cell shut-off, and translation of late mRNAs. Thus, the mutation in ONYX-015 compromises the ability of the mutant virus to reproduce itself in tumor cells. As such, it would be highly desirable to create an E1B-55K mutant that fails to bind and inactivate p53, yet is still capable of performing the late viral functions. Such a mutation would allow the virus to replicate selectively in cells that are deficient in p53 function without compromising the efficiency of the virus to replicate in tumor cells. The present invention fulfills these and other needs.
The references discussed herein are provided solely for their disclosure prior to the filing date of the present application. Nothing herein is to be construed as an admission that the inventors are not entitled to antedate such disclosure by virtue of prior invention.